CHARACTERIZATION OF DESERT DATE (Balanites aegyptiaca)

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identify some saponins from plant extracts (Liu et al., 2003; Liang et al., 2002; Cui et al., 2000). Balanites aegyptiaca is a desert plant species that is primarily grown in most of the arid regions of the world. Although it is considered to be the most adapted plant species of all dry arid and semiarid region plants, it is highly neglected. Literature study has revealed that various parts of this plant contain saponins, and fruit mesocarp contains the highest quantity. Studies show that most of the ethnobotanical uses of this plant are attributed to its saponin content. Saponin content in B. aegyptiaca has been reported in the literature. However, to the best of our knowledge, neither the saponin in B. aegyptiaca provenance grown in Israel nor the technique of using liquid chromatography-electrospray ionizationmass spectrometry (LC-ESI-MS) have been reported. This study describes the separation of methanol-extracted B. aegyptiaca mesocarp saponins by high-performance liquid chromatography-refractive index (HPLC-RI) to detect the whole spectrum of its major saponins, followed by electrospray ionization-mass spectrometry (ESI-MS) combined with multistage mass spectrometry (MS n ) to identify the major steroidal saponins of B. aegyptiaca grown in Israel. The structural determination of selected major saponins was also carried out using high-field NMR techniques. Material and Methods Plant material Fully ripened fruits of B. aegyptiaca were collected from B. aegyptiaca plants grown in Kibbutz Samar located in the Arava rift valley of southern Israel between the Dead Sea and Red Sea, close to the Jordanian border, 40 km north from the Rea Sea's Gulf of Eilat. The plant was botanically authenticated by Prof. Uzi Plitman from the herbarium in the Hebrew University of Jerusalem. Voucher specimen (76816) was deposited in the herbarium of the Hebrew University of Jerusalem. The epicarp (outer cover) was gently removed by hand and the mesocarp (pulp) was manually extracted with a knife. The mesocarps were first freeze-dried with a lyophilizer (Christ Alpha 1-4, Germany), then stored in an electric desiccator (Sanplatec Corp., Israel) for further use. 17
Extraction and separation For the extraction, the freeze-dried mesocarp was pulverized with a mortar and pestle, then combined with methanol (1:10) and shaken continuously overnight in a high-speed electric shaker (Tuttnauer, Jerusalem, Israel), followed by centrifugation at 3500 rpm for 18 min at 20ºC (Hermble, Germany) and the supernatant collected. Two subsequent extractions of the residue were carried out in methanol using only vortexes and centrifugation. After three successive extractions, the supernatant was clear. The supernatants were combined and the methanol was evaporated off in a rotary evaporator (Heta-Holten A/S, Denmark) under reduced temperature (below 40ºC). After dissolving the residue in H2O (1:2 w/v), the extract was defatted three times using a 1:4 ratio of n-hexane. The water was then removed from the defatted extract with a lyophilizer. The semicrystalline dried saponin extract (55% by weight of mesocarp) was designated ME (methanol extract) and kept refrigerated (4ºC) for further use. Liquid chromatography A Waters (Milford, MA, USA) 2690 HPLC system equipped with an auto sampler, a Luna 5u 100A C18 column (250 × 2 mm, 5 µm), an HPLC pump, and an Agilent (Palo Alto, CA, USA) G134A RI detector, was operated at room temperature. The mobile phase was 70:30 MeOH/H2O at a flow rate of 0.2 mL/min. The injection volume was 10 µL. In some experiments, the ME was eluted in methanol in solid phase extraction (SPE) cartridges (C18, 35 mL, 10 g), after discarding the first elution in water and then 35% methanol before injection. The methanol was again evaporated under reduced temperature and the extract was dissolved in methanol-water (70:30) and filtered (0.45 µm) before injection. Twenty-two percent by weight was obtained from the SPE elution. The evaporative light scattering (ELS) detector, a Sedex 75 (SEDERE, France), Luna C18 column (250 × 2.0 mm, 5 µm particle size; Phenomenex), equipped with a guard, was used for the separations, with the same isocratic mobile phase of 70:30 MeOH/H2O. A 100 µL sample was injected at a flow rate adjusted to 0.3 mL/min. The ELS detector separation was performed at ambient temperature. The ELS detector probe temperature was set at 50°C, the gain was set at 9, and the nitrogen nebulizer gas was adjusted to 2.0 bar. This 18
Page 1 and 2: CHARACTERIZATION OF DESERT DATE (Ba
Page 3 and 4: This work was carried out under the
Page 5 and 6: Abstract The desert date (Balanites
Page 7 and 8: of various Balanites tissues sugges
Page 9 and 10: Contents Acknowledgements i Abstrac
Page 11 and 12: Materials and methods 38 - Sample c
Page 13 and 14: - Effect of the saponin source on p
Page 15 and 16: Figure 2.9 1 H NMR spectrum showing
Page 17 and 18: List of Tables Table 2.1 Major HPLC
Page 19 and 20: Glossary CID collision induced diss
Page 21 and 22: Thesis organization This thesis is
Page 23 and 24: fruits have been found in Pharaohs'
Page 25 and 26: Fig 1.3. Map showing the Balanites
Page 27 and 28: themselves provide shade and shelte
Page 29 and 30: should be no surprise that the name
Page 31 and 32: Association with membranes Most bio
Page 33 and 34: the African or Indian samples. No w
Page 35 and 36: Objective and Hypothesis Objectives
Page 37: CHAPTER 2 Characterization of the f
Page 41 and 42: and analysed with an ELS detector (
Page 43 and 44: c d a Figure 2.2. Electrospray mass
Page 45 and 46: corresponds with the claims of earl
Page 47 and 48: Table 2.2. MS n Electrospray mass s
Page 49 and 50: 1209, and could be isomers of these
Page 51 and 52: compound as 26-(O-β-D-glucopyranos
Page 53 and 54: In the 1 H NMR spectrum, resonances
Page 55 and 56: Figure 2.11. Structure of the sapon
Page 57 and 58: Conclusions From this study we can
Page 59 and 60: that a study of the saponin profile
Page 61 and 62: HPLC pump and a refractive index (R
Page 63 and 64: Table 3.1 Peaks, retention time, am
Page 65 and 66: (a) (b) (c) (d) (e) Figure 3.2 ESI-
Page 67 and 68: Table 3.2 The LC-ESI-MS and corresp
Page 69 and 70: quoted here also are integers obtai
Page 71 and 72: (a) (b) (c) (d) (e) (f) (g) (i) Fig
Page 73 and 74: considered cleavage behavior, from
Page 75 and 76: Table 3.4 The LC-ESI-MS and corresp
Page 77 and 78: The results of this study also show
Page 79 and 80: ich extracts of Agave sisalana (Piz
Page 81 and 82: food preparation based on baby food
Page 83 and 84: Table 4.3 LC50 and LC90 (ppm) of sa
Page 85 and 86: 1 2 3 Figure 4.1 Structure of the t
Page 87 and 88: When the images of dead larvae were
Page 89 and 90:
CHAPTER 5 Antifungal activity of sa
Page 91 and 92:
visually and microscopically, and s
Page 93 and 94:
Effects of the SREs on in vitro gro
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Table 5.2 Effect of saponin rich ex
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(Table 5.3). In the case of C. cocc
Page 99 and 100:
CHAPTER 6 Effect of saponins on del
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astomatous cuticles from the upper
Page 103 and 104:
Results Effect of the saponin sourc
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Effect of humidity and temperature
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Table 6.3 Effect of humidity and te
Page 109 and 110:
Discussion and conclusions Agro-mat
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solutions used in our studies has s
Page 113 and 114:
CHAPTER 7 General conclusions and s
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as a delivery adjuvant, not only in
Page 117 and 118:
Botanic Gardens, Kew. 960 pp. Bushw
Page 119 and 120:
Ganzera, M., Bedir, E., Khan ,I.A.
Page 121 and 122:
Keukens, E.A.J., de Vrije, T., van
Page 123 and 124:
Morrissey, J.P., Osbourn, A.E. (199
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Schonherr, J., Riederer, M. (1986).
Page 127 and 128:
Appendices A1 - A4: Articles publis
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new and alternative sources of thes
Page 131 and 132:
first dissolved in 1 ml methanol an
Page 133 and 134:
In Table 1, the total sapogenin as
Page 135 and 136:
114 5. Beneytout JL, Nappez C, Lebo
Page 137 and 138:
Appendix: A2 Natural Product Commun
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min, and finally rinsed three times
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level was significantly increased f
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Table1. Effect of plant growth regu
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A B C D Fig 1. Different callus typ
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Appendix: A3 African Journal of Bio
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The data obtained were statisticall
Page 151 and 152:
Table 4. Mortality of Culex pipiens
Page 153 and 154:
Appendix: A4 Dengue Bulletin 2005;
Page 155 and 156:
of the Institutes for Applied Resea
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References 136 1. Halstead SB. Glob
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112 H20a-H21-H23b-H24bb 23a 31.5 1.
Page 161 and 162:
112.0 H15 β -H17a-H20a-H21 23a 31.
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112 H20b-H24aa-H24bb 23a 31.4 1.64
Page 165 and 166:
םהיתויוליעפו םהיתו
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. םיינגוזה םורדו תח
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םוקאזה ישרושב ירקי
Page 171:
םיטביה לש הנבהה היי
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CHARACTERIZATION OF DESERT DATE (Balanites aegyptiaca)

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